An important aspect of protein structure-function studies and enzyme-targeted biorational inhibitor design is an understanding of how chemical catalysis is occurring a the enzyme active site. The detection and identification of enzyme reaction intermediates provide key insights into the enzyme catalytic reaction pathway. Rapid chemical quench methods involving off-line detection have proven very useful in identifying enzyme reaction intermediates. However, a limitation of this approach involves the inability to detect and characterize enzyme intermediates that are labile under the chemical quenching conditions. We have developed and demonstrated the utility for the real-time detection and characterization of enzyme intermediates on the millisecond time scale using a novel approach of rapid mixing/time-resolved electrospray ionization (ESi) mass spectrometry (MS). The overall objective of the studies described in this proposal is to further develop and demonstrate the broad applicability of the rapid mix/time-resolved ESIMS approach for detecting and quantitating enzyme intermediates and defining enzyme reaction kinetic pathways. The following specific aims are designed to accomplish this objective: 1. Demonstrate the ability of rapid mixing/time-resolved ESI-MS to quantitatively determine kinetic parameters. 2. Demonstrate the broader utility of the rapid mixing/time-resolved ESI-MS to identify putative chemically labile enzyme intermediates. 3. Demonstrate the ability of rapid mixing/time-resolved ESI-MS to monitor specific phosphorylation and dephosphorylation events in protein signaling. These studies will aid in pioneering a powerful and novel analytical methodology for detecting, characterizing and quantitating enzyme intermediates on a rapid time scale.